An apical expression signal of the renal type IIc Na⁺-dependent phosphate cotransporter in renal epithelial cells

Abstract

ϩ -dependent phosphate cotransporter (NaPi-IIc) is specifically targeted to, and expressed on, the apical membrane of renal proximal tubular cells and mediates phosphate transport. In the present study, we investigated the signals that determine apical expression of NaPi-IIc with a focus on the role of the N-and the C-terminal tails of mouse NaPi-IIc in renal epithelial cells [opossum kidney (OK) and Madin-Darby canine kidney cells]. Wild-type NaPi-IIc, the cotransporter NaPi-IIa, as well as several IIa-IIc chimeras and deletion mutants, were fused to enhanced green fluorescent protein (EGFP), and their cellular localization was analyzed in polarized renal epithelial cells by confocal microscopy and by cell-surface biotinylation. Fluorescent EGFP-fused NaPi-IIc transporter proteins are correctly expressed in the apical membrane of OK cells. The apical expression of N-terminal deletion mutants (deletion of N-terminal 25, 50, or 69 amino acids) was not affected by truncation. In contrast, C-terminal deletion mutants (deletion of C-terminal 45, 50, or 62 amino acids) did not have correct apical expression. A more detailed mutational analysis indicated that a domain (amino acids WLHSL) in the cytoplasmic C terminus is required for apical expression of NaPi-IIc in renal epithelial cells. We conclude that targeting of NaPi-IIc to the apical cell surface is regulated by a unique amino acid motif in the cytoplasmic C-terminal domain.Npt2c; SLC34A3; localization; OK cell; hereditary hypophosphatemic rickets with hypercalciuria INORGANIC PHOSPHATE (P i ) reabsorption in renal proximal tubules is required for P i homeostasis in the body. Na ϩ -dependent P i transporters (NaPi) in the brush-border membrane (BBM) of proximal tubule cells mediate the rate-limiting step in the overall P i -reabsorption process (26,28). Type IIa (NaPiIIa) and type IIc (NaPi-IIc) NaPi cotransporters are expressed in the apical membrane of proximal tubule cells and mediate P i transport (27, 38). The extent of P i reabsorption in the proximal tubules is determined largely by the abundance of the NaPi-IIa cotransporter (11,28,29).Several groups have shown that hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a Mendelian disorder of renal P i reabsorption (39, 42), results from the lack of a functional NaPi-IIc protein, leading to severe renal wasting with hypophosphatemia (5,16,19,22,25). These data suggest that the NaPi-IIc cotransporter plays an important role in renal P i reabsorption and may be a key determinant of plasma P i concentrations in humans. However, it is not clear why a loss of function of the less abundant and energetically less favorable electroneutral NaPi-IIc transporter causes hypophosphatemia/rickets and osteomalacia in humans, whereas mutations in the more abundant electrogenic NaPi-IIa elicits a mild skeletal phenotype (3).The plasma membrane of epithelial cells is divided into two separate membrane compartments, the apical and the basolateral domains (36,37). This polarity is maintained by intracellular mach...

Hepatectomy-Related Hypophosphatemia

“…Affinity-purified anti-NaPi-IIa, 40 anti-NaPi-IIc, 41 antiNaPi-IIb (for rat BBMVs), 42 and anti-NaPi-4, 43 were described previously. Anti-NaPi-IIb (for mouse BBMVs; Alpha Diagnostics, San Antonio, TX) 44 and anti-Nampt (for rat kidney total lysate or total cell lysate of OK cells; AdipoGen, Incheon, Korea) were used following the manufacturers' instructions.…”

“…The cells were grown at 37°C in a humidified environment (5% CO 2 and 95% air). 43 Before transfection, the cells were plated in six-well dishes. Subconfluent cultures were transfected with 4 ml LipofectAmine2000 (Invitrogen) and 4 mg plasmids according to the manufacturer's instructions.…”

“…Preparation of total cell lysates and crude membranes was performed as previously described. 43,48 Biotinylated cell surface proteins were isolated using the Pierce cell surface protein isolation kit (Pierce, Rockford, IL) according to the manufacturer's instructions and as previously reported with some modifications. 43,48 Immunostaining of OK Cells OK cells were immunostained as previously described.…”

“…43,48 Biotinylated cell surface proteins were isolated using the Pierce cell surface protein isolation kit (Pierce, Rockford, IL) according to the manufacturer's instructions and as previously reported with some modifications. 43,48 Immunostaining of OK Cells OK cells were immunostained as previously described. 43 OK cells, grown to subconfluence on glass coverslips, and were transfected with constructs expressing rat Nampt cDNA fused with C terminus His or V5 tags.…”

“…43,48 Immunostaining of OK Cells OK cells were immunostained as previously described. 43 OK cells, grown to subconfluence on glass coverslips, and were transfected with constructs expressing rat Nampt cDNA fused with C terminus His or V5 tags. The cells were fixed with 3% paraformaldehyde and permeabilized with 0.05% saponin in PBS.…”